This invention relates to a process for producing alpha-isopropylmalic acid by fermentation. More particularly, it relates to a process for producing alpha-isopropylmalic acid by cultivating a new strain of the yeast Yarrowia lipolytica in an aqueous medium containing assimilable sources of carbon, nitrogen, L-leucine and inorganic salts under submerged aerobic conditions and to an improved process for recovering alpha-isopropylmalic acid from fermentation broth.
Isolation of alpha-isopropylmalic acid from culture medium of a leucine-requiring mutant of Neurospora crassa is reported by Burns et al., Biochemistry 2, 1053 (1963) and by Calvo et al., Biochemistry 3, 2024 (1964).
The accumulation of alpha-isopropylmalic acid [beta-carboxy-beta-hydroxyisocaproic acid or S(-)-2-hydroxy-2-(1-methylethyl)butanedioic acid] as a metabolic product of leucine-requiring mutants (auxotrophs) of Saccharomyces cerevisiae and its isolation from fermentation broths containing it by acidification and ether extraction of the broth is reported by Sai, Agr. Biol. Chem. 32, 522-4, (1968).
A partial bibliography of the discovery of alpha-isopropylmalic acid as a precursor for leucine biosynthesis in Torulopsis utilis, as a metabolic product of some strains of bacteria and molds which requires leucine for growth, and its enzymatic formation in cell-free preparations of baker's yeast is presented by Sai (loc. cit.).
French Pat. No. 2,417,548, granted Oct. 19, 1979, describes preparation of alpha-isopropylmalic acid by aerobic fermentation of Brevibacterium or Corynebacterium species.
Fultz et al., J. Bacteriol. 142, 513-520 (1980) report construction of two Salmonella typhimurium strains by a series of P22-mediated transductions. One strain accumulated and excreted alpha-isopropylmalic acid exclusively, the amount of said acid isolatable from culture media thereof being more than five times that produced by Neurospora crassa. The second accumulated and excreted a mixture of alpha- and beta-isopropylmalic acids.
Alpha-isopropylmalic acid is reported by Mikami et al., Agr. Biol. Chem. 34 (6), 977-9 (1970) to be a plant growth regulator. Japanese Pat. No. 70,032,919 of Oct. 23, 1970 (Chem. Abstr. 75, 31511v, 1971) describes its use for improving the flavor of tobacco. Its use as an intermediate for preparation of the tobacco flavor modifiers 2-isopropyl-4,4-dimethyl-5-methylene-2-cyclopenten-1-one and 2,5-diisopropyl-2-cylcopenten-1-one by dry distillation is disclosed in Japanese Kokai 77,113,945, published Sept. 24, 1977 (Chem. Abstr. 88, 62045u, 1978). German Offenlegungsschrift No. 2,922,222, published Dec. 13, 1979 (Chem. Abstr. 92, 152039g, 1980) describes its use and that of its sodium and ammonium salts as setting retardants for gypsum.